Using a novel infection assay for M. tuberculosis, we have shown that iron is a necessary co-factor with cytokines to restrict the spread of M. tuberculosis through human monocyte monolayers. TNF is felt to be one of the key cytokines in granuloma formation. TNF is a powerful monocyte chemoattract and induces monocyte aggregation. Iron-mediated restriction of M. tuberculosis is associated with decreased monocyte aggregation and marked downmodulation of TNF secretion relative to activated monocytes not receiving iron. The overall objective of this proposal is to further delineate the role of iron, TNF and other cytokines in events leading to granuloma formation and restriction of M. tuberculosis infection. SPECIFIC AIMS of this proposal are as follows: AIM 1: To further refine an in vitro model of events leading to early granuloma formation. AIM 2: To delineate the effect of the addition of cytokines in various sequences on events leading to early granuloma formation and restriction of M. tuberculosis growth. AIM 3: To determine the effect of blocking and promoting TNF secretion and/or activity on M. tuberculosis growth in cytokine-treated monocyte monolayers. -AIM 4: To determine whether TNF receptor modulation on monocytes affects M. tuberculosis growth in monocyte monolayers. AIM 5: To determine the relative roles of extracellular uptake and cell to cell spread in growth of M. tuberculosis in cytokine-treated monocyte monolayers. AIM 6: To determine the composite effect of the iron counterregulatory agents IFN gamma/calcitriol and TNF on the iron metabolism of human monocytes. Understanding the role of TNF in M. tuberculosis infection could lead to interventions designed to decrease inappropriate or excessive TNF secretion with drugs such as thalidomide. The knowledge gained from experiments in this proposal may lead to an enhanced understanding of the clinical setting in which such drugs might be beneficial in mycobacterial disease. To achieve the AIMS of this proposal, tissue culture assays specifiCally developed to study the role of M. tuberculosis infections will be used. These assays allow for the study of the cell to cell interactions of mononuclear phagocytes yielding insights into events leading to granuloma formation. The assays will be used to study TNF-iron interactions in growth restriction of M. tuberculosis in human monocytes focusing on cellular morphology and M. tuberculosis cell to cell spread.